Stem cells treated by in vitro fucosylation and methods of use

ABSTRACT

A method of in vitro fucosylation of selectin ligands on cord blood-derived hematopoietic stem cells for bone marrow transplantation is disclosed. In this method, an effective amount of an α1,3-fucosyltransferase, e.g., α1,3-fucosyltransferase VI, is used in vitro to treat cord blood-derived hematopoietic stem cells to convert non-functional PSGL-1 or other ligands on the cell surface into functional forms that bind selectins, especially P-selectin or E-selectin. The treated cells have enhanced effectiveness in reconstituting bone marrow in patients in need of such therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 12/707,481,filed Feb. 17, 2010, now U.S. Pat. No. 8,084,255, which is acontinuation of U.S. Ser. No. 11/448,359, filed Jun. 7, 2006, now U.S.Pat. No. 7,776,591, issued Aug. 17, 2010, which is a continuation ofU.S. Ser. No. 10/769,686, filed Jan. 30, 2004, now U.S. Pat. No.7,332,334, issued Feb. 19, 2008, which claims the benefit under 35U.S.C. 119(e) of U.S. Provisional Application Ser. No. 60/463,788, filedApr. 18, 2003, each of which is hereby expressly incorporated byreference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant 5P5OHL54502awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

This invention generally relates to methods of treating hematopoieticstem cells (HSCs) for improving their therapeutic usefulness and moreparticularly, but not limited to, treating hematopoietic stem cellsderived from cord blood, and hematopoietic stem cells thus treated.

During inflammation, P-selectin and E-selectin cooperatively mediateleukocyte rolling and adhesion on vascular surfaces (McEver, R. P.Selectins: lectins that initiate cell adhesion under flow. Curr OpinCell Biol. 2002 October; 14:581-856). In the process of bone marrowtransplantation, P-selectin and E-selectin also mediate the homing ofintravenously injected HSCs to bone marrow. (Frenette, P. S., Subbarao,S., Mazo, I. B., Von Andrian, U. H., Wagner, D. D. Endothelial selectinsand vascular cell adhesion molecule-1 promote hematopoietic progenitorhoming to bone marrow. Proc. Natl. Acad. Sci. USA. 1998;95:14423-14428). In most tissues, P-selectin and E-selectin areexpressed on endothelial cells after stimulation of agonists, but theyare expressed constitutively on bone marrow endothelial cells. Selectinsuse α2,3-sialylated and α1,3-fucosylated glycans such as sialylLewis^(x) (sLe^(x)) on glycoproteins or glycolipids as ligands.P-selectin binds to the N-terminal region of P-selectin glycoproteinligand-1 (PSGL-1), which contains tyrosine sulfates and an O-glycancapped with sLe^(x). E-selectin binds to one or more different sites onPSGL-1. To interact with E-selectin, PSGL-1 does not require tyrosinesulfation, but expression of sLe^(x) on O-glycans enhances binding.E-selectin also interacts with other ligands on HSCs. An isoform of CD44on HSCs has been shown to bind to E-selectin in vitro (Dimitroff, C. J.,Lee, J. Y., Rafii, S., Fuhlbrigge, R. C., Sackstein, R. CD44 is a majorE-selectin ligand on human hematopoietic progenitor cells. J. Cell Biol.Jun. 11, 2001; 153:1277-1286). Another potential ligand for E-selectinon HSCs is E-selectin ligand-1 (ESL-1) (Wild, M. K., Huang, M. C.,Schulze-Horsel, U., van Der Merwe, P. A., Vestweber, D. Affinity,kinetics, and thermodynamics of E-selectin binding to E-selectinligand-1. J Biol. Chem. 2001 Aug. 24; 276:31602-31612). Each of theseglycoprotein ligands is thought to carry sLe^(x) structures.

Hematopoietic stem cells harvested from one individual can betransplanted to the bone marrow of another individual following anintravenous infusion. The approach has been widely used in treatment ofvarious hematological disorders such as leukemia (Thomas, E. D. History,current results, and research in marrow transplantation. PerspectivesBiol. Med. 38:230-237.1995). Clinically, human HSCs are obtained fromthree different sources: bone marrow, adult peripheral blood aftermobilization, and cord blood obtained from umbilical cords afterdelivery. Although there are more than 5 million unrelated bone marrowvolunteer donors registered worldwide, finding a fully human leukocyteantigen (HLA)-matched unrelated donor remains a problem for manypatients because of HLA polymorphism. Compared with bone marrow andadult peripheral blood, cord blood has several potential advantages, inparticular the wide and rapid availability of cells and less stringentrequirements for HLA identity between donor and recipient because of thelower risk of acute and chronic graft-versus-host disease (GVHD) (Rocha,V., et. al., Comparison of outcomes of unrelated bone marrow andumbilical cord blood transplants in children with acute leukemia. Blood.97:2962-71.2001). Potential advantages of transplantation using cordblood HSCs rather than HSCs from bone marrow or adult peripheral bloodinclude: (1) a large potential donor pool; (2) rapid availability, sincethe cord blood has been prescreened and tested; (3) greater racialdiversity can be attained in the banks by focusing collection efforts onhospitals where children of under represented ethnic backgrounds areborn; (4) reduced risk or discomfort for the donor; (5) rarecontamination by viruses; and (6) lower risk of graft-versus-hostdisease (wherein the donor's cells attack the patient's organs andtissues), even for recipients with a less-than-perfect tissue match.Thus, cord blood-derived HSCs have been increasingly used for bonemarrow transplantation in recent years.

In the transplantation setting, the intravenously infused HSCsspecifically extravasate in the bone marrow to engraft and proliferate,a process that is defined as HSC homing. Homing has been studiedextensively both in vivo and in vitro and is believed to rely onadhesion molecule interactions between HSCs and endothelium of bonemarrow. Selectins are a group of adhesion molecules containing aN-terminal carbohydrate-recognition domain related to those inCa⁺⁺-dependent (C-type) animal lectins. P-selectin, expressed onactivated platelets and endothelial cells, and E-selectin, expressed onactivated endothelial cells, bind to glycoconjugate ligands onleukocytes and HSCs. The best-characterized glycoprotein ligand isPSGL-1, a mucin with many sialylated and fucosylated O-linkedoligosaccharides. PSGL-1 is expressed on leukocytes and HSCs. Studieswith PSGL-1-deficient mice have shown that PSGL-1 mediates leukocytetethering to and rolling on P-selectin and supports tethering toE-selectin in flow. PSGL-1 also binds to L-selectin, which initiatesleukocyte-leukocyte interactions that amplify leukocyte rolling oninflamed endothelial cell surfaces. In human PSGL-1, the P-selectin andL-selectin binding site comprises a peptide sequence containing threetyrosine sulfate residues near a threonine to which is attached aspecific branched, fucosylated core-2 O-glycan (McEver, R. P., Cummings,R. D. Role of PSGL-1 binding to selectins in leukocyte recruitment. 3Clin Invest. 100:S97-103. 1997; R. P. McEver: Selectins: Ligands thatinitiate cell adhesion under flow. Curr. Op. in Cell Biol. 14: 581-586,2002, which discloses that CD34, glycosylated cell adhesion molecule-1(GlyCAM-1) and podacalyxin are selectin ligands for L-selectin). Thefucose moiety is essential for P-selectin binding as measured by invitro assays using synthetic glycosulfopeptides. The fucosylation iscatalyzed by a family of α1,3-fucosyltransferases. Among them,α1,3-fucosyltransferase IV (FT-IV) and α1,3-fucosyltransferase VII(FT-VII) are primarily expressed in human leukocytes. These enzymescatalyze the transfer of a fucose residue from a donor, e.g.,GDP-fucose, to an acceptor in α1,3-linkage to GlcNAc inGal-GlcNAc-sequences. Both FT-IV and FT-VII make the fucose additionwhich is necessary to form the sLe^(x) structure(NeuAcα2,3Galβ1,4-[Fucα1,3]GlcNAcβ1-R). The sLe^(x) on a core-2 O-glycanattached to a specific threonine in the N-terminal amino acid sequenceof human PSGL-1 is critical for binding to P-selectin.

HSCs have the potential to differentiate into different lineages ofhematopoietic cells such as red blood cells, myeloid cells, lymphocytesand platelets. Human HSCs express a surface glycoprotein, CD34, which isroutinely used for HSC identification and separation. Such human CD34⁺cells (cells which express CD34 antigen) represent a heterogeneouspopulation of progenitors with various degrees of hematopoieticmaturation. The absence of (“−”) or reduced (“low”) expression ofanother surface protein, CD38, on human CD34⁺ cells is considered to bea surrogate marker of a primitive subpopulation of CD34⁺ cells. Thus,the cells of the CD34⁺CD38^(low/−) sub-population, which compriseapproximately 10-20% of the total CD34⁺ cells from bone marrow or adultperipheral blood, are highly enriched for multiprogenitor and stem cellactivity, including engraftment ability. Notably, approximately 30% ofcord blood HSCs are CD34⁺CD38^(low/−). However, unlike CD34⁺CD38^(low/−)adult peripheral blood stem cells, cord blood CD34⁺CD38^(low/−) HSCs areknown to have reduced homing to murine bone marrow, which is primarilydependent on interactions of human HSCs with murine P-selectin on themicrovascular endothelium (Hidalgo, A., Weiss, L. A., and Frenette, P.S. Functional selectin ligands mediating human CD34⁺ cell interactionwith bone marrow endothelium are enhanced postnatally. Adhesion pathwaysmediating hematopoietic progenitor cell homing to bone marrow. J. Clin.Invest. 110:559-569. 2002). Flow cytometry analyses indicate that thishoming defect results from non-functional PSGL-1 expressed on theseCD34⁺CD38^(low/−) cord-blood derived HSCs. Thus, the impaired ability ofthe CD34⁺CD38^(low/−) HSCs to bind to P-selectin explains in at least inpart the delayed platelet and myeloid engraftment associated with cordblood HSC transplantation. The use of cord blood HSCs fortransplantation has been primarily restricted to children (which requirefewer cells for transplantation) due to the limited quantities anddefective homing ability of HSCs isolated from umbilical cords.

An invention which corrects the homing defect of HSCs wouldsignificantly increase the potential of umbilical cord blood as a sourceof hematopoietic stem cells and would thereby lead to lower risks foracute and chronic graft-versus-host disease and improved success of bonemarrow reconstitution.

SUMMARY OF THE INVENTION

The present invention in one embodiment contemplates a method oftreating HSCs comprising the steps of providing a quantity or populationof HSCs, at least some of which lack or have reduced expression ofsurface protein CD38, and treating the quantity or population of HSCs invitro with an α1,3 fucosyltransferase and a fucose donor, wherein thetreated HSCs have enhanced binding to P-selectin and E-selectin.Furthermore, the HSCs are typically characterized as comprisingP-selectin glycoprotein ligand-1 (PSGL-1) and/or other selectin ligandswhich does not effectively bind to P-selectin or E-selectin. Moreparticularly, the PSGL-1 or other selectin ligands which occurs on theCD34⁺ CD38^(low/−) HSCs lack, or have fewer, fucosylated glycans,particularly O-glycans, and may for example, have PSGL-1 which havecore-2 O-glycans which comprise NeuAcα2,3Galβ1,4GlcNAc and which lackfucose in α1,3 linkage to the GlcNAc. The HSCs, in their untreated stateprior to fucosylation as described herein, have reduced bone marrowhoming ability. In one embodiment of the invention, the HSCs are derivedfrom human umbilical cord blood, though they may be derived from bonemarrow or peripheral blood, as long as they are characterized as havingenhanced bone marrow homing ability after the fucosylation treatment. Inthe method contemplated herein, the α1,3 fucosyltransferase may be, forexample, an α1,3 fucosyltransferase IV, an α1,3 fucosyltransferase VI,or an α1,3 fucosyltransferase VII, or a combination thereof. The fucosedonor may be, for example, GDP-fucose.

The invention further contemplates in one embodiment a composition oftreated human HSCs which comprise cord blood-derived CD34⁺ HSCs lackingor having reduced expression of surface protein CD38 (CD38^(low/−)),wherein the HSCs are able to bind to P-selectin or E-selectin. The HSCsmay be disposed in a pharmaceutically acceptable carrier, or diluent, orvehicle for storage or administration to a patient. The invention isfurther directed to a treatment method, comprising administering aneffective amount of the HSCs to a subject having a hematologicaldisorder or other disease requiring or benefiting from a transplantationof HSCs for treatment.

As noted above, after the fucosylation treatment described herein, thetreated CD34⁺ HSCs (including CD34⁺CD38^(low/−) HSCs) have enhancedbinding to P-selectin or E-selectin, as compared to untreated CD34⁺HSCs. Enhanced binding to P-selectin (or E-selectin) is defined as atleast 10% of the treated HSCs having fluorescence in a P-selectin (orE-selectin, respectively) binding assay which is greater than apredetermined fluorescence threshold (as defined below). In anotherembodiment, at least 25% of the treated HSCs exceed the predeterminedfluorescence threshold. In another embodiment, at least 50% of thetreated HSCs exceed the predetermined fluorescence threshold. In anotherembodiment, at least 75% of the treated HSCs exceed the predeterminedfluorescence threshold. In another embodiment, at least 90% of thetreated HSCs exceed the predetermined fluorescence threshold. In anotherembodiment, at least 95% of the treated HSCs exceed the predeterminedfluorescence threshold.

The present invention further contemplates a blood product produced bythe method including the steps of providing a quantity or population ofHSCs, at least a portion of which are CD34⁺ and which lack or havereduced expression of protein CD38, and treating the quantity of HSCs invitro with an α1,3 fucosyltransferase and a fucose donor, wherein themajority of the treated HSCs have enhanced binding to P-selectin (orE-selectin) as described herein. The quantity of HSCs are preferablyderived from umbilical cord blood but may be obtained from bone marrowor adult peripheral blood. The quantity or population of HSCs couldcomprise a portion, or unfractionated sample, of blood or bone marrow.

BRIEF DESCRIPTION OF THE FIGURES

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. A. CD34 antibody staining of mononuclear cells (MNCs) isolatedfrom human cord blood. B. CD34 antibody staining of cells afterCD34-enrichment. C. Isotope control IgG staining of CD34⁺ cells. Axesare fluorescence intensity as measured by flow cytometry.

FIG. 2. A. CD34⁺ cells isolated from cord blood express PSGL-1. B. CD34⁺cells consist of about 30% CD34⁺CD38^(low/−) cells (primitiveprogenitors) and about 65% CD34⁺CD38⁺ cells. Axes are fluorescenceintensity as measured by flow cytometry.

FIG. 3. A. CD34⁺ cells are gated as P-selectin binding cells (R2) ornon-P-selectin binding cells (R1). B. 24%±5% of CD34⁺ cells from R1region have no or reduced expression of CD38. The result isrepresentative of four independent flow cytometry analyses and showsthat significant numbers of non-P-selectin binding HSCs are CD34⁺ andCD38^(low/−).

FIG. 4. Viability of cells after in vitro fucosylation as measured bypropidium iodide (PI) staining. A. Cells without treatment. B.Sham-treated cells. C. FT-VI-treated cells. Axes are fluorescenceintensity as measured by flow cytometry.

FIG. 5. A. 15% of the CD34⁺ cells obtained from cord blood express lowor no fucosylated epitopes as stained with sLe^(x)-specific monoclonalantibody HECA 452. B. In vitro α1,3-fucosylation with FT-VI andGDP-fucose dramatically increases sLe^(x) epitopes on cord blood-derivedCD34⁺ cells. Axes are fluorescence intensity as measured by flowcytometry.

FIG. 6. Titration of soluble P-selectin binding to CD34⁺ HSCs by flowcytometry for determining a saturating amount of P-selectin.

FIG. 7. Binding of a saturable concentration of soluble P-selectin tocord blood-derived CD34⁺ cells. A. About 27% of untreated cordblood-derived CD34⁺ cells do not bind to or have low level binding toP-selectin. B. In vitro α1,3-fucosylation converts CD34⁺ cells which arenegative or low for P-selectin binding into cells which are positive andhigh for P-selectin binding. C. and D. P-selectin binds to PSGL-1 oncord blood-derived CD34⁺ cells as verified by blocking monoclonalantibodies to P-selectin (G1) and PSGL-1 (PL1). EDTA also inhibitsbinding, consistent with the requirement for Ca²⁺ to support P-selectinbinding to PSGL-1. Axes are fluorescence intensity as measured by flowcytometry.

FIG. 8. Rolling of CD34⁺ cells on human serum albumin (HSA) or on humanP-selectin under shear force. Treatment of cord blood-derived CD34⁺cells with GDP-fucose and FT-VI significantly augments cell rolling onP-selectin in shear flow.

FIG. 9. Binding of a saturable concentration of soluble E-selectin tocord blood-derived CD34⁺ cells. A. About 24% of untreated cordblood-derived CD34⁺ cells do not bind to or have low level binding toE-selectin. B. In vitro α1,3-fucosylation converts CD34⁺ cells which arenegative or low for E-selectin binding into cells which are positive andhigh for E-selectin binding. C. and D. E-selectin binds to cordblood-derived CD34⁺ cells as verified by blocking monoclonal antibodiesto E-selectin (9A9). EDTA also inhibits binding. Axes are fluorescenceintensity as measured by flow cytometry. The result is representative ofthree independent measurements.

FIG. 10. In vitro fucosylation significantly augments CD34⁺ cellsrolling on human soluble E-selectin under shear forces. A and B.Treatment of CB CD34⁺ cells with GDP-fucose and FT-VI significantlyenhances the number of cells rolling on E-selectin under different shearforces. The rolling is E-selectin dependent as the cells did not roll onhuman serum albumin (HSA) and rolling was specifically blocked by ES1, amAb to human E-selectin, but not by PL1, a mAb which binds to theP-selectin binding site of PSGL-1. C and D. The fucosylated CD34⁺ cellsare more resistant to shear forces and roll slower than untreated CD34⁺cells. The data represent the mean±SD of four independent experiments.

FIG. 11. Fucosylated CB HSCs exhibit enhanced engraftment in bone marrowof sublethally irradiated NOD/SCID mice. Bone marrow (BM) or peripheralblood (PB) from mice 6 weeks after transplantation with 8×10⁶sham-treated or FTVI-treated CB cells were analyzed for engraftment ofhuman-derived hematopoietic cells. (A) Flow cytometry analysis of BM andPB cells stained with a mAb to the human pan-leukocyte marker CD45demonstrated a doubling of human-derived cells in mice transplanted withfucosylated CB cells. (B) Compared with mice transplanted with CB cellswithout fucosylation, BM cells from mice transplanted with fucosylatedCB cells contain significantly more human colony-forming progenitors,which include BFU-E, CFU-GM, and CFU-GEMM, as demonstrated by humanhematopoietic progenitor assays. Bone marrow of control mice injectedwith saline only produced no colonies, confirming the specificity of theassay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention in one embodiment contemplates a method oftreating HSCs comprising providing a quantity or population of HSCswhich lack or have reduced expression (less than the normal level ofexpression of CD38) of surface protein CD38, and treating the quantityor population of HSCs in vitro with an α1,3 fucosyltransferase and afucose donor, wherein the HSCs so treated have enhanced binding toP-selectin or E-selectin over the untreated HSCs. Furthermore, theuntreated HSCs are typically characterized as predominantly comprisingPSGL-1 or other selectin ligands which do not adequately bind toP-selectin or E-selectin. The PSGL-1 or other selectin ligands whichoccur on the CD38^(low/−) HSCs lack or have reduced numbers offucosylated glycans, such as O-glycans, and may for example, have PSGL-1which have core-2 O-glycans which comprise NeuAcα2,3Galβ1,4GlcNAc butwhich lack a fucose in α1,3 linkage to the GlcNAc. The CD38^(low/−)HSCs, in their untreated state prior to fucosylation, have reduced bonemarrow homing ability. Preferably, the HSCs are derived from humanumbilical cord blood (CB), although they may be derived from bone marrowor peripheral blood, as long as they are characterized as needing, orbenefiting from, further fucosylation to enhance their bone marrowhoming ability. In the method contemplated herein, the α1,3fucosyltransferase may be for example α1,3 fucosyltransferase IV, α1,3fucosyltransferase VI, or α1,3 fucosyltransferase VII. The fucose donormay be for example GDP-fucose.

The invention contemplates in one embodiment a composition of treatedhuman HSCs which comprise cord blood-derived HSCs lacking or havingreduced expression of surface protein CD38 (CD38^(low/−)), wherein thetreated HSCs comprise PSGL-1 or other selectin ligands that are properlyfucosylated (e.g., comprises sialyl Lewis^(x)) and which are able tobind to P-selectin (or E-selectin). The treated HSCs may be disposed ina pharmaceutically acceptable carrier or vehicle for storage oradministration to a patient. The invention is further directed to atreatment method, comprising administering an effective amount of thetreated HSCs to a subject having a hematological disorder or otherdisease requiring transplantation of HSCs for treatment.

In one embodiment, the composition of treated HSCs comprises apopulation of human HSCs derived from umbilical cord blood, at least aportion of which are characterized as CD34⁺CD38^(low/−) HSCs havingenhanced binding to P-selectin (or E-selectin). Enhanced binding toP-selectin (or E-selectin) is defined as at least 10% of the treatedHSCs having fluorescence in a P-selectin binding assay (or E-selectinbinding assay, respectively) which is greater than a predeterminedfluorescence threshold. In another embodiment, at least 25% of thetreated HSCs exceed the predetermined fluorescence threshold. In anotherembodiment, at least 50% of the treated HSCs exceed the predeterminedfluorescence threshold. In another embodiment, at least 75% of thetreated HSCs exceed the predetermined fluorescence threshold. In anotherembodiment, at least 90% of the treated HSCs exceed the predeterminedfluorescence threshold. In another embodiment, at least 95% of thetreated HSCs exceed the predetermined fluorescence threshold. Thecomposition of human HSCs preferably is disposed in apharmaceutically-acceptable carrier or vehicle for storage or foradministration to a subject.

The predetermined fluorescence threshold in one embodiment is determinedby first obtaining a sample of cells containing at least 100CD34⁺CD38^(low/−) HSCs from a mononuclear fraction of ordinary umbilicalcord blood (cord blood from healthy full term babies). This control(baseline) sample of HSCs is assayed using the P-selectin binding assay(or E-selectin binding assay) described elsewhere herein, or by anyother P-selectin fluorescence binding assay (or E-selectin bindingassay, respectively) known in the art. P-selectin (or E-selectin)binding fluorescence levels are measured for the CD34⁺CD38^(low/−) HSCsin the control (baseline) sample. In one embodiment, a fluorescencevalue is selected which exceeds the P-selectin (or E-selectin) bindingfluorescence levels of at least 95% of the CD34⁺CD38^(low/−) HSCs in thecontrol sample. The selected fluorescence value is designated as thepredetermined fluorescence threshold against which is compared theP-selectin (or E-selectin) binding fluorescence of the treated (i.e.,fucosylated) HSCs.

The present invention further contemplates a blood product produced bythe method of providing a quantity or population of HSCs, at least aportion of which are CD34⁺ and which lack or have reduced expression ofprotein CD38, and treating the quantity of HSCs in vitro with an α1,3fucosyltransferase and a fucose donor, wherein the majority of thetreated HSCs bind to P-selectin (or E-selectin). The quantity of HSCsmay be derived from umbilical cord blood, but may be obtained from bonemarrow or adult peripheral blood.

In general, the present invention contemplates a method whereinnon-functional or suboptimally functional PSGL-1 or other selectinligands expressed on cells, including umbilical cord cells, bone marrowcells, or blood cells, including HSCs, are modified by in vitroα1,3-fucosylation technology, e.g., to correct the homing defect, whichimproves their use in bone marrow transplantation.

As noted above, CD34⁺ cord blood HSCs may be defined as either CD38⁺(positive for CD38) or CD38^(low/−) (reduced or no expression of CD38).CD38^(low/−) cord blood HSCs can be identified using fluorescencetechniques as described below. Cord blood HSCs are treated with aCD34-binding antibody having a fluorophore linked thereto, and with aCD38-binding antibody having a different fluorophore linked thereto.CD34⁺ cells are defined as those HSCs which exhibit fluorescence fromthe anti-CD34 antibody fluorophore upon irradiation. CD38^(low/−) HSCsare defined as the 30% of CD34⁺ HSCs which have the lowest fluorescenceas measured from the anti-CD38 binding antibody, or as the CD34⁺ HSCswhich have anti-CD38 binding antibody fluorescence levels of 50 units orless (as measured by a fluorescence flow cytometer as describedelsewhere herein). In one embodiment, the anti-CD34 binding antibodyfluorophore is FITC (fluorescein isothiocyonate) while the anti-CD38binding antibody fluorophore is phycoerythrin (PE).

As explained previously, CD34⁺ cells express PSGL-1 or other selectinligands, yet a significant amount of primitive CD34⁺ cells which are lowin or lack CD38, (e.g., which comprise about 30% of the total CD34⁺ cordblood cells), do not bind to P-selectin (or E-selectin) or bind only lowamounts of P-selectin (or E-selectin, respectively). PSGL-1 is ahomodimeric mucin expressed on almost all leukocytes including CD34⁺cells. To be functional, i.e., able to bind to P-selectin or E-selectin,PSGL-1 requires several post-translational modifications leading toformation of an sLe^(x) group thereon, including α1,3-fucosylation.Insufficient α1,3-fucosylation, for example, results in impaired abilityof naive T cells to interact with vascular selectins. In the presentinvention it has been discovered that the inability of cord bloodderived HSCs to bind to P-selectin or E-selectin is due to inadequateα1,3-fucosylation of PSGL-1 or other selectin ligands. Therefore, thebasis of the present invention is that the treatment of CD34⁺ cells invitro with an α1,3-fucosyltransferase (e.g., FT-VI), which alsocatalyzes the synthesis of the sLe^(x) structure, will increasefucosylation of PSGL-1 or other selectin ligands and for example, tothereby correct the homing defect of the HSCs.

Fucosyltransferases which are able to transfer fucose in α1,3 linkage toGlcNAc are well known in the art. Several are available commercially,for example from Calbiochem. Further, at least five different types ofα1,3 fucosyltransferases (FTIII-VII) are encoded by the human genome.These include: the Lewis enzyme (FTIII), which can transfer fucoseeither a (1,3) or a (1,4) to Galβ4GlcNAc or Galβ3GlcNAc respectively(Kukowska-Latallo et al., Genes Dev. 4:1288, 1990); FTIV, which forms a(1,3) linkages, which does not prefer sialylated precursors (Goelz, etal., Cell 63; 1349, 1989; Lowe, et al., J. Biol. Chem. 266; 17467,1991); FTV (Weston, et al., J. Biol. Chem. 267:4152, 1992a) and FTVI(Weston, et al., J. Biol. Chem. 267:24575, 1992b) which form α(1,3)linkages, which can fucosylate either sialylated or nonsialylatedprecursors, and FTVII, (Sasaki, et al., J. Biol. Chem. 269:14730, 1994);Natsuka, et al., J. Biol. Chem. 269:16789, 1994) which can fucosylateonly sialylated precursors.

FTIII is encoded by GDB:135717; FTIV by GDB:131563; FTV by GDB:131644;FTVI by GDB:135180; and FTVII by GDB:373982. A sixth α1,3fucosyltransferase (FTIV) is encoded by GDB:9958145 (Genome DatabaseAccession ID numbers are available from the GDB™ Human Genome DatabaseToronto (Ontario, Canada): The Hospital for Sick Children, Baltimore(Md., USA): Johns Hopkins University, 1990. Available from Internet: URLhttp://www.gdb.org/). The present invention further contemplates usingother, non-human α1,3 fucosyltransferases available and known to thoseof ordinary skill in the art, for example as shown in U.S. Pat. Nos.6,399,337 and 6,461,835 which are hereby expressly incorporated byreference herein in their entireties.

As noted previously, human HSCs can be obtained for treatment with α1,3fucosyltransferase, for example, by separation from the other cells in asource of umbilical cord blood, peripheral blood, or bone marrow.Various techniques may be employed to separately obtain theCD34⁺CD38^(low/−) stem cells alone, or in combination with CD34⁺CD38⁺HSCs. Monoclonal antibodies are particularly useful for identifyingmarkers (surface membrane proteins) associated with particular celllineages and/or stages of differentiation. The antibodies may beattached to a solid support to allow for crude separation. Theseparation techniques employed should maximize the retention ofviability of the fraction to be collected. The particular techniqueemployed will depend upon efficiency of separation, cytotoxicity of themethodology, ease and speed of performance, and necessity forsophisticated equipment and/or technical skill.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads, and “panning” with antibody attached toa solid matrix, e.g., plate, or other convenient technique. Techniquesproviding accurate separation include fluorescence activated cellsorters, which can have varying degrees of sophistication, e.g., aplurality of color channels, low angle and obtuse light scatteringdetecting channels, and impedance channels.

Conveniently, the antibodies may be conjugated with markers, such asmagnetic beads, which allow for direct separation; biotin, which can beremoved with avidin or streptavidin bound to a support; fluorochromes,which can be used with a fluorescence activated cell sorter (FACS), orthe like, to allow for ease of separation of the particular cell type.Any technique may be employed which is not unduly detrimental to theviability of the remaining cells.

In one embodiment, the HSCs lacking the mature cell markers, may besubstantially enriched, wherein the cells may then be separated by theFACS or other methodology having high specificity. Multi-color analysesmay be employed with the FACS which is particularly convenient. Thecells may be separated on the basis of the level of staining for theparticular antigens. Fluorochromes, which may find use in a multi-coloranalysis, include phycobiliproteins, e.g., phycoerythrin andallophycocyanins, fluorescein, and Texas red, for example.Alternatively, HSCs can be treated with fucosyltransferases beforeseparation of the desired HSCs from the unfractionated blood or bonemarrow sample, for example, using total mononuclear cells from cordblood, peripheral blood, or bone marrow.

In one embodiment, the CD34⁺HSC, including CD34⁺CD38^(low/−) cells maybe treated by adding free fucosyltransferase to the cell composition,wherein the final blood product containing the fucosylatedCD34⁺CD38^(low/−) also contains the fucosyltransferase which was used totreat the cells. In another embodiment, the HSCs may be treated usingfucosyltransferases which are bound to a support, such as magneticbeads, or any other support known by those of ordinary skill in the art,which can be separated from the cell composition after the treatmentprocess is complete.

Methods and Results

Umbilical cord blood samples were obtained from normal full-term vaginaldeliveries in accordance with a protocol approved by the InstitutionalReview Board of the Oklahoma Medical Research Foundation (OMRF). 70 to100 ml of cord blood was collected per delivery. Sodium citrate was usedas anticoagulant. Any appropriate method known in the art for collectingcord blood is suitable, such as the method shown in U.S. Pat. No.6,440,010, which is expressly incorporated herein by reference in itsentirety. The CD34⁺ cells in the supernatant of the blood sample wereenriched with a CD34-isolation mini-MACS kit (Miltenyi Biotec, BergischGladbach, Germany). Cord blood was first mixed with an equal volume of6% dextran 70 in 0.9% sodium chloride (McGaw, Inc., Irvine, Calif.).After sedimentation of two to three hours, the cells in the supernatantwere removed, and washed once in Hanks' balanced salt solution (HBSS,Cellgro) containing 2 mM EDTA and 0.5% human serum albumin (HSA).Contaminating red blood cells were lysed in FACS Lysing solution (BDBiosciences, San Jose, Calif.). Low-density mononuclear cells (MNCs)were separated after centrifugation at 250 g over Ficoll-Hypaque(d=1.077 g/ml). CD34⁺ cells were purified from the MNC fraction usingthe CD34-isolation mini-MACS kit following the manufacturer'sinstructions. The purity of the isolated CD34⁺ cells was about 96% asexamined by flow cytometry (FIG. 1). The following experiments were thencarried out.

Verification by Flow Cytometry that CD34⁺ Cells Isolated from Cord BloodExpress PSGL-1 and the CD34⁺ Cells are Heterogeneous.

For this purpose, triple-colored staining was used. The cells enrichedby the mini-MACS sorting were incubated with anti-CD34 monoclonalantibody (mAb, clone AC136 from Miltenyi Biotec) conjugated with FITC,anti-CD38 mAb conjugated with PE (BD Pharmingen, San Diego, Calif.), andanti-PSGL-1 monoclonal antibody conjugated with Cy5 (BD Pharmingen, SanDiego, Calif.) after blocking the Fc receptor with human IgG. Afterwashing, the cells were analyzed by flow cytometry on a FACScan (BectonDickinson). Data were collected using the CellQuest program. Lightscatter-gated events were plotted on a log scale of fluorescenceintensity. Virtually all CD34⁺ cells express PSGL-1 (FIG. 2A), and about30% of the CD34⁺ cells have low or no expression of CD38 (FIG. 2B),representing the sub-population of primitive progenitor cells. Further,about 25% of the HSCs that do not bind to P-selectin are CD34⁺ andCD38^(low/−) (FIG. 3). These results confirm existing data.

In Vitro α1-3-Fucosylation of PSGL-1 on Purified CD34⁺ Cells.

To introduce fucose on core 2 O-glycans attached to PSGL-1 or otherselectin ligands on CD34⁺ cells, 2-4×10⁶ cells were treated with 1 mMguanosine diphosphate (GDP)-fucose (Calbiochem), 20 mU/mLα1-3-fucosyltransferase VI (FT-VI) (Calbiochem), and 10 mM MnCl₂ in 0.5mL HBSS/1% HSA for 40 minutes at 37° C., in an atmosphere containing 5%CO₂. This treatment produces optimal fucosylation of PSGL-1 on CD34⁺cells as measured by maximum P-selectin binding, yet results in minimumtoxicity to CD34⁺ cells as tested by propidium iodide staining (FIG. 4).

Measurement of Fucosylated Epitopes on CD34⁺ Cells and Verification byFlow Cytometry that In Vitro α1,3-Fucosylation Creates FucosylatedEpitopes on CD34⁺ Cells.

Sialyl Lewis^(x) is a fucosylation epitope. By incubating with ananti-sLe^(x) mAb HECA 452 (rat IgM, hybridoma from American Type CultureCollection [ATCC]), we examined the sLe^(x) epitopes on the CD34⁺ cells.The bound mAb was detected with FITC-conjugated goat F(ab)′2 fragmentsto rat IgM (Caltag). As indicated by FIG. 5A, 26% of the CD34⁺ cellsobtained from cord blood express low or no fucosylated epitopes. Thesedata demonstrate that a subset of CD34⁺ cells are not properlyfucosylated. To investigate if in vitro α1,3-fucosylation can createfucosylated epitopes on the CD34⁺ cells, we stained the cells with HECA452 after treatment of the CD34⁺ cells with FT-VI and GDP-fucose in thepresence of Mn²⁺ using the method described above. We found that the invitro α1,3-fucosylation dramatically increased sLe^(x) epitopes on cordblood-derived CD34⁺ cells as indicated by HECA 452 staining (FIG. 5B).

P-Selectin Binding—Results

Verification of the Binding Profiles of Soluble P-Selectin on CordBlood-Derived CD34⁺ Cells.

For the P-selectin binding assay, cord blood-derived CD34⁺ cells, afterFc receptor blocking, were incubated with anti-CD34-PE and withP-selectin isolated from human platelets. P-selectin binding wasdetected with FITC-labeled S12, a non-blocking mAb to human P-selectin.Incubations of the cells were performed at 4° C. for 20 min. Asaturating amount of P-selectin was used in the experiments after aserial titration (FIG. 6). In control experiments, P-selectinincubations of the cells were carried out in the presence of G1, ablocking mAb to P-selectin, PL1, a blocking mAb to PSGL-1, or 10 mMEDTA, which eliminates Ca²⁺-dependent selectin-ligand interactions. Flowcytometry analyses showed that about 27% of the CD34⁺ cells (primarilycomprising the CD38^(low/−) cells) did not bind to P-selectin, which isconsistent with previously published data (FIG. 7A) (Hidalgo, A., Weiss,L. A., and Frenette, P. S. Functional selectin ligands mediating humanCD34⁺ cell interaction with bone marrow endothelium are enhancedpostnatally. Adhesion pathways mediating hematopoietic progenitor cellhoming to bone marrow. J. Clin. Invest. 110:559-569. 2002). FIG. 7Cshowed that P-selectin bound specifically to PSGL-1 on the CD34⁺ cellsbecause the G1 and PL1 antibodies and EDTA abolished binding.

Demonstration by Flow Cytometry that In Vitro α1,3-Fucosylation of theSurface of CD34⁺ Cells Increases Binding to P-Selectin.

The cord blood-derived CD34⁺ cells were first treated with GDP-fucoseand FT-VI as described above, and then stained with both anti-CD34-PEand P-selectin. The P-selectin binding was detected with FITC-labeledmAb S12. Treatment with exogenous FT-VI significantly increased bindingof CD34⁺ cells to human P-selectin (FIG. 7B). The augmented binding toP-selectin was due to the increased functional PSGL-1 on the CD34⁺ cellsafter the α1,3-fucosylation because binding was blocked by antibodies G1and PL1 and by EDTA (FIG. 7D). To find optimal conditions for in vitroα1,3-fucosylation, various incubation times and concentrations of FT-VI,GDP-fucose, and Mn were examined (data not shown). A condition (shownabove) was chosen for all the experiments that produced optimalfucosylation of PSGL-1 on CD34⁺ cells as measured by maximum P-selectinbinding (FIG. 7B), yet resulted in minimum toxicity to CD34⁺ cells astested by propidium iodide staining (FIG. 4).

Demonstration that In Vitro α1,3-Fucosylation Increases CD34⁺ CellAdhesion to P-Selectin in Physiological Shear Flow.

Cord blood-derived CD34⁺ cells were divided into two groups for furtherprocessing. One group was incubated with GDP-fucose and FT-VI asdescribed above, and another was treated with FT-VI without GDP-fucose(sham-treated control). The P-selectin-binding ability of the two groupsof cells was compared using an in vitro flow chamber rolling assaysystem as described below. P-selectin isolated from human platelets wasimmobilized in a parallel-plate flow chamber. A P-selectin site densityof 145 sites/μm² was used as measured by binding of ¹²⁵I-labeledanti-P-selectin mAb S12. Sham-treated or FTVI-treated CD34⁺ cells(10⁶/ml in Hanks' balanced salt solution and 0.5% human albumin) wereperfused over P-selectin at a wall shear stress of 1 dyn/cm². Theaccumulated number of rolling cells was measured with a videomicroscopysystem coupled to an image analysis system. The CD34⁺ cells rolled in aCa⁺⁺-dependent manner by human P-selectin-PSGL-1 interactions becauseEDTA and antibodies G1 and PL1 abolished the rolling, and no rolling wasobserved on plates coated only with human serum albumin (FIG. 8).Compared to sham-treated CD34⁺ cells, about 3-fold more FT-VI-treatedCD34⁺ cells rolled on P-selectin.

E-selectin Binding—Results

Binding Profiles of Soluble E-Selectin to CB-Derived HSCs.

Murine soluble E-selectin/human IgM chimera (E-selectin/IgM) was usedfor the fluid phase E-selectin binding assay. CD45/human IgM chimera wasused as negative control. The cells were incubated with theE-selectin/IgM after Fc receptor blocking. E-selectin binding was thendetected with FITC-labeled goat anti-human IgM polyclonal antibodies.The cells were also stained with PE-labeled anti-CD34 mAb (BDPharmingen, San Diego, Calif.). Incubations were performed at 4° C. for20 min. A saturated amount of E-selectin was used in the experimentsafter a serial titration. In control experiments, stainings were carriedout in the presence of 9A9, a blocking mAb to E-selectin, or 10 mM EDTA,which eliminates Ca²⁺-dependent selectin-ligand interactions. Flowcytometry analyses showed that about 25% of the CD34⁺ HSCs did not bindto E-selectin (FIG. 9A). FIG. 9C showed that the interaction of CD34⁺HSCs with E-selectin was specific because mAb 9A9 and EDTA abolished it.

In Vitro α1,3-Fucosylation Increases CD34⁺ HSC Binding to E-Selectin asMeasured by Flow Cytometry.

The CB-derived CD34⁺ HSCs were divided into two groups. One group(2-4×10⁶ cells) was incubated with 1 mM GDP-fucose, 20 mU/ml FTVI(Calbiochem), and 10 mM MnCl₂ in 0.5 ml HBSS/1% HSA for 40 minutes at37° C., in an incubator containing 5% CO₂. Another group was incubatedwith FT-VI without GDP-fucose (sham-treated control). The cells werethen stained with both anti-CD34 and E-selectin/IgM. After the exogenousα1,3-fucosyltransferase treatment, the binding of CD34⁺ HSCs toE-selectin increased from 75% to 95% (FIGS. 9A and B). The augmentedbinding to E-selectin was specific as verified by mAb 9A9 and EDTA (FIG.9D). The residual binding after Ab 9A9 and EDTA blocking seen in FIGS.9C and D was non-specific because cells stained with negative controlCD45/IgM had a similar profile (data not shown).

In Vitro α1,3-Fucosylation Increases HSC Adhesion to E-Selectin UnderPhysiological Shear Forces

The HSCs were divided into two groups and fucosylated as describedabove. The E-selectin-binding ability of the two groups of cells wascompared using an in vitro flow chamber rolling system. Briefly, solublehuman E-selectin was immobilized in a parallel-plate flow chamber. AnE-selectin site density of 200 sites/μm² was used as measured by bindingof ¹²⁵I-labeled anti-human E-selectin mAb ES1. Sham-treated orFT-VI-treated HSCs (10⁶/ml in HBSS and 0.5% HSA) were perfused overE-selectin under different shear forces. The accumulated number andshear resistance of the rolling cells were measured with avideomicroscopy system coupled to an image analysis system. At shearforces examined, about 2-3 times more FT-VI-treated HSCs rolled onE-selectin compared to the sham-treated HSCs (FIGS. 10A and B). TheFT-VI-treated cells also rolled with lower velocity and were moreresistant to detachment by shear forces (FIGS. 10C and D). Theinteraction of HSCs with E-selectin was specific, as mAb ES1 abolishedrolling and rolling was not observed on plates coated only with HSA(FIG. 10B). PL1, which blocks binding of P-selectin to PSGL-1, did notaffect HSC rolling on E-selectin (FIG. 10B), confirming that E-selectinmediates rolling by binding to other sites on PSGL-1 or to othercell-surface ligands.

These results indicate that in vitro α1,3-fucosylation enhancesphysiologically-relevant rolling adhesion of CD34⁺ cells to P-selectinand E-selectin under flow.

In Vivo Example

Fucosylated HSCs exhibit enhanced engraftment in bone marrow in vivo.

Methods

By in vitro analyses, it has been demonstrated herein that CB HSCstreated with GDP-fucose and FTVI exhibited a significant increase influid-phase binding to P-selectin and E-selectin and rolled much betteron P-selectin and E-selectin coated surfaces under different wall shearforces, compared with CB HSCs without fucosylation. The fucosylated CBHSCs are further shown herein to have improved homing to and engraftmentin bone marrow in vivo. Nonobese diabetic severe combinedimmunodeficiency (NOD/SCID) mice have been well established asxenogeneic recipients for in vivo studies of human HSCs. We havecompared human hematopoietic engraftment in NOD/SCID mice transplantedwith CB HSCs with or without fucosylation.

Male and female pathogen-free (NOD/SCID) mice (The Jackson Laboratory),4-5 weeks of ages, were used as recipients. The mice were irradiated(230 rad) 2 or 3 hours before intravenous injections of FTVI-treated(fucosylated) or sham-treated (treated with FTVI but without GDP-fucose)CB HSCs (8×10⁶/mouse in 300 μl saline) respectively. Control mice eachreceived 300 μl saline without CB HSCs.

Six weeks after transplantation, the mice were bled and sacrificed. Bonemarrow cells were isolated from both femora and filtered through a100-mm mesh filter to remove debris. After lysis of red blood cells, thebone marrow nucleated cells from each mouse were resuspended in HBSS ata concentration of 1×10⁶/ml. The engraftment was analyzed by both flowcytometry and human hematopoietic progenitor assays. For flow cytometry,bone marrow nucleated cells were incubated with a Cy5-conjugatedanti-human CD45 mAb (BD Pharmingen, San Diego, Calif.).

For human hematopoietic progenitor assays, 1×10⁵ bone marrow nucleatedcells per 35-mm culture dish were plated into MethoCult H4433 media(Stem Cell Technologyies, Vancouver, Canada) in duplicate and incubatedat 37° C., 5% CO₂. Total colonies, burst-forming units-erythroid(BFU-E), colony-forming units-granulocyte/macrophage (CFU-GM), andcolony-forming units-granulocyte/megakaryocyte/macrophage (CFU-GEMM)were counted on day 14 of culture and analyzed. The human origin of thecolonies was confirmed by flow cytometry analysis of cells collectedfrom different colonies stained with mAbs to human CD45 for myeloidcells and glycophorin A for erythroid cells, respectively.

Results

The irradiated NOS/SCID mice that received fucosylated CB HSCs had 2-3fold more CD45 positive human-derived hematopoietic cells in bone marrowand peripheral blood than mice that received sham-treated CB HSCs, asanalyzed by flow cytometry (FIG. 11A). The significantly improvedengraftment of human hematopoietic progenitors in bone marrow of micetransplanted with fucosylated cells was multilineage as demonstrated bythe increases of BFU-Es, CFU-GMs, and CFU-GEMMs (FIG. 11B). Of note, thesizes of the colonies derived from CB HSCs were not significantlydifferent in either recipient group (data not shown), indicating thatfucosylation did not change the growth potential of the CB progenitors.Thus, the in vivo study demonstrates that the FTVI-treated CB HSCs havemuch higher potential to home to and engraft in bone marrow of NOD/SCIDmice than the sham-treated cells do. These results show that the HSCs ofthe present invention will have enhanced bone marrow engraftment inhumans.

Utility

The fucosylated HSCs described herein may be used in a variety of ways.For example, since the cells are naive (primitive), they can be used tofully reconstitute the bone marrow of an irradiated subject and/or anindividual subjected to chemotherapy.

Among the conditions which can be treated by administration ofhemopoietic stem cells according to the present invention are leukemiasand lymphomas such as chronic myelocytic (myelogenous) leukemia (CML),juvenile chronic myelogenous leukemia (JCML), acute myelocytic leukemia(AML), acute lymphocytic leukemia (ALL), malignant lymphoma, multiplemyeloma, aplastic anemia gravis, myelodysplastic syndrome (MDS), andautoimmune diseases, for example.

Other diseases that may be treated with the treated HSCs of the presentinvention are: Gunther's disease, Hunter syndrome, Hurler syndrome,neuroblastoma, non-Hodgkin's lymphoma, Wiskott-Aldrich syndrome,X-linked lympho-proliferative syndrome, and solid tissue tumors, such asbreast cancer.

In these treatments, populations of these treated HSCs can be given to apatient whose marrow has been destroyed by ablative therapy.

The cells of the present invention can be administered by intravenousinjection, for example, or by any other appropriate method known bythose of ordinary skill in the art. In methods for treating a hostafflicted with a disease or condition, a therapeutically effectiveamount of HSCs is that amount sufficient to reduce or eliminate thesymptoms or effects of the disease or condition. The therapeuticallyeffective amount administered to a host will be determined on anindividual basis and will be based, at least in part, on considerationof the individual's size, the severity of symptoms to be treated, andthe results sought. Thus, a therapeutically effective amount can bedetermined by one of ordinary skill in the art of employing suchpractice in using no more than routine experimentation. For detailedinformation on HSC transplantations, “Hemopoietic Stem CellTransplantation, Its Foundation and Clinical Practice” [Modern Medicine,Special Issue, 53, 2, 1998] can be consulted and the descriptions giventhere are expressly incorporated herein by reference in their entirety.

In preparing the dosage of fucosylated stem cells to be administered, avariety of pharmaceutically acceptable carriers can be utilized. Thecarrier, diluent or vehicle may contain a buffering agent to obtain aphysiologically acceptable pH, such as phosphate-buffered saline, and/orother substances which are physiologically acceptable and/or are safefor use. In general, the material for intravenous injection in humansshould conform to regulations established by the Food and DrugAdministration, which are available to those in the field.Pharmaceutically-acceptable carriers may be combined, for example, in a1 volume:1 volume ratio, with the treated HSC composition. The carriermay be for example, M199 or RPMI 1640 medium. Furthermore, in preparingsaid dosage form, various infusions in common use today can also beemployed. In one embodiment, the dose amount conventionally used in thetransplantation of HSCs can be employed. The dosage may be, for example,about 0.01-10×10⁸ treated MNCs/kg of weight (which includes treatedCD38^(low/−) HSCs or other treated HSCs as defined elsewhere herein) ofthe patient, or more, or less where appropriate.

As described herein, the present invention contemplates a method oftreating HSCs, comprising providing a quantity of HSCs, at least aportion of the HSCs lacking or having reduced expression of surfaceprotein CD38, and treating the quantity of HSCs in vitro with anα1,3-fucosyltransferase and a fucose donor forming treated HSCs, whereinthe treated HSCs have enhanced binding to P-selectin or E-selectin. Inone embodiment, the portion of HSCs lacking or having reduced expressionof surface protein CD38 has reduced bone marrow homing ability. The HSCsmay be derived from human umbilical cord blood, and may be anunfractionated quantity of human umbilical cord blood. Alternatively,the HSCs may be derived from peripheral blood, and may be anunfractionated quantity of peripheral blood. Alternatively, the HSCs maybe derived from bone marrow, and may be an unfractionated quantity ofbone marrow. The portion of HSCs lacking or having reduced expression ofsurface protein CD38 comprises PSGL-1 or other structures which haveunfucosylated glycans or unfucosylated O-glycans. In the present method,the portion of HSCs lacking or having reduced expression of surfaceprotein CD38 may comprise PSGL-1 having core-2 O-glycans comprisingNeuAcα2,3 Gal β1,4 GlcNAc and which are absent a fucose in α1,3 linkageto the GlcNAc or which comprise other glycans which lack properfucosylation. In one embodiment, at least 50% of the treated HSCs haveP-selectin binding fluorescence which exceeds a predeterminedfluorescence threshold in a P-selectin binding assay or E-selectinbinding fluorescence which exceeds a predetermined fluorescencethreshold in an E-selectin binding assay (as described elsewhereherein). In the present method, the α1,3 fucosyltransferase may be α1,3fucosyltransferase IV, α1,3 fucosyltransferase VI, or α1,3fucosyltransferase VII. Further, the fucose donor may be GDP-fucose.

The present invention further contemplates a composition of HSCs whichcomprises CD34⁺ HSCs derived from umbilical cord blood and lacking orhaving reduced expression of surface protein CD38, wherein at least 10%of the CD34⁺ HSCs bind to P-selectin (or E-selectin), and apharmaceutically-acceptable carrier. In the composition, in alternativeembodiments, at least 25%, 50%, 75%, 90%, or 95% of the CD34⁺ HSCs bindto P-selectin (or E-selectin).

The present invention also contemplates treating a subject with ahematological disease or other condition requiring a transplantation ofHSCs by administering a quantity of the composition of treated HSCsdescribed herein to the subject having a hematological disease or othercondition requiring a transplantation of HSCs. The hematological diseasemay be, for example, acute lymphocytic leukemia, acute myelogenousleukemia, myelodispasia, chronic myelogenous leukemia, juvenile chronicmyelogenous leukemia, or sickle cell anemia.

Furthermore, the present invention contemplates a blood productcomprising a population of human HSCs comprising cells characterized asCD34⁺CD38^(low/−), wherein at least 10% of the CD34⁺CD38^(low/−) HSCsbind to P-selectin or E-selectin. In the blood product, in alternativeembodiments, at least 25%, 50%, 75%, 90%, or 95% (or any percentageinclusive) of the CD34⁺CD38^(low/−) HSCs bind to P-selectin orE-selectin. In the blood product, the human HSCs may be derived fromhuman umbilical cord blood, adult peripheral blood, or bone marrow. Theblood product may also comprise a pharmaceutically acceptable carrier orvehicle, and may also comprise a free fucosyltransferase or afucosyltransferase bound to a support.

The present invention also contemplates a blood product produced by themethod comprising providing a quantity of HSCs, at least a portion ofthe HSCs lacking or having reduced expression of surface protein CD38,and treating the quantity of HSCs in vitro with anα1,3-fucosyltransferase and a fucose donor to produce treated HSCs,wherein at least 10% of the treated HSCs bind to P-selectin orE-selectin. In an alternative embodiment at least 25%, 50%, 75%, 90%, or95% (or any percentage inclusive) of the treated HSCs of the bloodproduct bind to P-selectin or E-selectin. In the blood product, thequantity of HSCs may be derived from human umbilical cord blood,peripheral blood, or bone marrow.

While the invention has been described above in connection with variousembodiments so that aspects thereof may be more fully understood andappreciated, it is not intended to limit the invention to theseparticular embodiments. On the contrary, it is intended to cover allalternatives, modifications and equivalents as may be included withinthe scope of the invention as defined by the appended claims. Thus theprevious examples will serve to illustrate the practice of thisinvention, it being understood that the particulars shown are by way ofexample and for purposes of illustrative discussion of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description ofprocedures as well as of the principles and conceptual aspects of theinvention.

All references, patents and patent applications cited herein are herebyincorporated herein by reference in their entireties.

Changes may be made in the construction and the operation of the variouscompositions and products described herein or in the steps or thesequence of steps of the methods described herein without departing fromthe spirit and scope of the invention as described herein.

What is claimed is:
 1. A process of producing a population of cellsexhibiting enhanced binding to a selectin, the process comprisingcontacting in vitro on the surface of the cells a glycoprotein, whichwhen fucosylated is a selectin ligand, with an α1,3 fucosyltransferasein the presence of a fucose donor so as to fucosylate the glycoproteinand thereby produce a population of fucosylated cells exhibitingenhanced binding to the selectin, wherein the selectin ligand isP-selectin glycoprotein ligand-1 (PSGL-1), CD44, E-selectin ligand-1(ESL-1), CD34, glycosylated cell adhesion molecule-1 (GlyCAM-1), orpodacalyxin.
 2. The process of claim 1, wherein the selectin ligand isP-selectin glycoprotein ligand-1 (PSGL-1).
 3. The process of claim 1,wherein the selectin ligand is CD44.
 4. The process of claim 1, whereinthe selectin ligand is E-selectin ligand-1 (ESL-1).
 5. The process ofclaim 1, wherein the selectin ligand is CD34.
 6. The process of claim 1,wherein the selectin ligand is glycosylated cell adhesion molecule-1(GlyCAM-1).
 7. The process of claim 1, wherein the selectin ligand ispodacalyxin.
 8. The process of claim 1, wherein the cells are bloodcells.
 9. The process of claim 8, wherein the blood cells are peripheralblood cells.
 10. The process of claim 1, wherein the cells are umbilicalcord cells.
 11. The process of claim 1, wherein the cells are bonemarrow cells.
 12. The process of claim 1, wherein the fucose donor isGDP-fucose.
 13. The process of claim 1, wherein the α1,3fucosyltransferase is α1,3-fucosyltransferase III (FTIII).
 14. Theprocess of claim 1, wherein the α1,3 fucosyltransferase isα1,3-fucosyltransferase IV (FTIV).
 15. The process of claim 1, whereinthe α1,3 fucosyltransferase is α1,3-fucosyltransferase V (FV).
 16. Theprocess of claim 1, wherein the α1,3 fucosyltransferase isα1,3-fucosyltransferase VI (FTVI).
 17. The process of claim 1, whereinthe α1,3 fucosyltransferase is α1,3-fucosyltransferase VII (FTVII). 18.A process of producing a population of cells exhibiting enhanced bindingto a selectin, the process comprising contacting in vitro on the surfaceof the cells a glycoprotein, which when fucosylated is a selectinligand, with an α1,3 fucosyltransferase in the presence of a fucosedonor so as to fucosylate the glycoprotein and thereby produce apopulation of fucosylated cells exhibiting enhanced binding to theselectin, wherein the selectin ligand comprises P-selectin glycoproteinligand-1 (PSGL-1).
 19. A process of producing a population of cellsexhibiting enhanced binding to a selectin, the process comprisingcontacting in vitro on the surface of the cells a glycoprotein, whichwhen fucosylated is a selectin ligand, with an α1,3 fucosyltransferasein the presence of a fucose donor so as to fucosylate the glycoproteinand thereby produce a population of fucosylated cells exhibitingenhanced binding to the selectin, wherein the selectin ligand comprisesCD44.
 20. A process of producing a population of cells exhibitingenhanced binding to a selectin, the process comprising contacting invitro on the surface of the cells a glycoprotein, which when fucosylatedis a selectin ligand, with an α1,3 fucosyltransferase in the presence ofa fucose donor so as to fucosylate the glycoprotein and thereby producea population of fucosylated cells exhibiting enhanced binding to theselectin, wherein the selectin ligand comprises E-selectin ligand-1(ESL-1).
 21. A process of producing a population of cells exhibitingenhanced binding to a selectin, the process comprising contacting invitro on the surface of the cells a glycoprotein, which when fucosylatedis a selectin ligand, with an α1,3 fucosyltransferase in the presence ofa fucose donor so as to fucosylate the glycoprotein and thereby producea population of fucosylated cells exhibiting enhanced binding to theselectin, wherein the selectin ligand comprises CD34.
 22. A process ofproducing a population of cells exhibiting enhanced binding to aselectin, the process comprising contacting in vitro on the surface ofthe cells a glycoprotein, which when fucosylated is a selectin ligand,with an α1,3 fucosyltransferase in the presence of a fucose donor so asto fucosylate the glycoprotein and thereby produce a population offucosylated cells exhibiting enhanced binding to the selectin, whereinthe selectin ligand comprises glycosylated cell adhesion molecule-1(GlyCAM-1).
 23. A process of producing a population of cells exhibitingenhanced binding to a selectin, the process comprising contacting invitro on the surface of the cells a glycoprotein, which when fucosylatedis a selectin ligand, with an α1,3 fucosyltransferase in the presence ofa fucose donor so as to fucosylate the glycoprotein and thereby producea population of fucosylated cells exhibiting enhanced binding to theselectin, wherein the selectin ligand comprises podacalyxin.